Electrochromatography ("EC") is one of many variations of the electrophoresis separation technique. In EC, retention of solute by some form of stationary retentive phase provides the selectivity for separation as is the case for a normal chromatographic separation. However, in EC, the fluid-mediated transport of solute is through electroosmotic flow, which is provided by the support material that holds the retentive phase. The interest in EC stems from the belief that zone broadening is generally smaller because the flow profile is uniform, and that flow can be achieved with smaller particles. Uniform flow profiles and smaller particles lead to higher resolution, which is very desirable in complex analysis or in situations where the zone width can be compromised to run at faster analysis time. This is in contrast to the parabolic flow profile found in pressure-driven flow from a pump-driven packed bed chromatographic system. In pressure-driven systems, small particles can cause huge pressure drops in the packed bed which leads to pump fatigue and shorter column lifetime.
In EC, like other techniques that work by electrophoresis and/or electroosmosis, a current is sent through a column to enact transport of molecules. This current is proportional to the cross-sectional area of the column through which transport takes place. Capillary-sized columns are preferred in EC because a low cross-sectional column area produces the lowest amount of heat (which can destroy the molecules to be separated, and reduce the separation efficiency due to formation of viscosity gradients), and further because the high surface area to volume ratio of capillaries allows heat to be dissipated at a faster rate as compared with larger sized columns.
The EC technique has been implemented in a number of ways in capillary systems. For example, Yan, C., et al. Anal. Chem. 1995, 67, 2026-2029 describe the use of capillaries packed with small particles. This technique requires the capillary to be packed with a solid phase such as beads, which is difficult to do in a capillary, particularly achieving uniform density. Furthermore, supporting frits are required to hold the beads in place. These frits are difficult to make and often generate air bubbles during the separation.
Capillaries used in the open-tube configuration are described in Bruin, G. J. M., et al., J. Chrom. 1990, 517, 557-572. The disadvantages of this technique include the lack of abundant retentive phase per column length and small performance increase as compared with pressure-driven flow using a micro LC column.
The packed capillary configuration has recently been augmented by new retentive phases. In situ polymerized sol-gel media are described by Guo, Y., Colon, L. A. Anal. Chem. 1995, 67, 2511-2516; in situ polymerized polyacrylamide gel media are described by Fujimoto, C. et al., Anal. Chem. 1996, 68,2753-2757; and in situ polymerized hydrophobic alkyl (meth)acrylate gel media are described in U.S. Pat. No. 5,647,979 to Liao et al. These gel media have various levels of chromatographic performance and require different levels of expertise for preparation. Furthermore, these preparations are inconvenient and impractical. The in situ polymerization takes a substantial amount of time, and subsequent purification must also be done in situ--taking yet more substantial time. The preparation of in situ materials makes purification extremely difficult due to mass transport limitations of diffusion and convection in porous materials. Thus, EC baseline stability suffers in these gel media systems.
In addition, because these gel systems are prepared in situ, their use depends on the ease of cleaning the freshly prepared capillary prior to stable analytical utilization. Because the pore space is small in these systems, they are susceptible to clogging. Once clogged, the lifetime of the capillary is reached and a new capillary must be used. Irreversible adsorption will reduce the electroosmotic velocity for this type of EC. This is contrasted to normal pressure-driven flow LC where the driving velocity is not affected by irreversible adsorption.
Accordingly, a need exists to provide for the convenient preparation, purification, use and re-use of EC columns.